Membrane Proteins

Membranes and their proteins

Every cell is enclosed by a plasma membrane . In addition, the
cytoplasm of eukaryotic cells contains a variety of organelles, each bounded
by an internal membrane .
All biological membranes are assemblies of lipid and protein molecules held
together by non-covalent interactions.

The lipids are amphipathic , ie they have a hydrophilic polar 'head' and a
hydrophobic non-polar 'tail', and they therefore spontaneously form bilayers in
aqueous solution.

The membrane is fluid, with individual lipid molecules able to diffuse freely
within the bilayer. It may therefore be thought of as a '2-dimensional solvent'
for the proteins. Proteins diffuse 100 - 100,000 times slower than the lipids.
There are three major types of lipids in membranes:
phospholipids (the most abundant), glycolipids and cholesterol.

The composition of membranes varies depending on function.
The plasma membranes of animal cells are approximately 50% protein in terms of
weight. The inner mitochondrial membrane, which is involved in energy
transduction, is about 75% protein, while the figure for myelin, which functions
as an insulator, is roughly 25%.
Many membrane proteins, termed
glycoproteins, have covalently bonded carbohydrate groups. In mammalian
cells such chains are often attached to an
Asparagine's
amide group. Carbohydrate
chains occur on the extracellular side of plasma membranes, and the luminal side
of intracellular membranes around organelles.

Membrane function

A membrane is not simply a physical barrier
between the cell cytoplasm and its external environment, or between different
compartments of a cell. The functions of membranes depend on the proteins
within them. Some of these proteins have largely a structural role, while many
enzymes are associated with membranes.

some membrane proteins are structural links between the cell's cytoskeleton
and the extracellular matrix

other proteins form assemblies in the membrane which join neighbouring cells
in a tissue

some membrane proteins selectively transport specific molecules and ions into or out
of the cell or organelle

others are receptors for chemical signals from outside the cell/organelle

some proteins catalyze reactions in specialized membranes, such as the
production of energy from sunlight, and the coupling of the flow of electrons
to ATP synthesis.

Categories of membrane proteins

The manner in which a protein associates with a membrane depends on its structure
and can be categorized as follows:

The protein is partially inserted into the membrane. Like the lipids it is of
amphipathic nature: it has a domain where hydrophobic residues are exposed on the
surface, which is in contact with the inner, hydrophobic part of the lipid bilayer,
and a polar domain which interacts with the solvent and with the polar heads of the
lipids. Some of the G-proteins, which bind guanyl nucleotides belong to this
group. Few examples have been found in which such a protein occurs in the
extracellular side of the plasma membrane, although mellitin,
the bee-venom toxin, may be one. (back to figure)

Mellitin 2mlt (46Kb) [Bbk|BNL|ExP|Waw|Hal]
Use this short script to show the hydrophobic and polar
faces.

More commonly, there is a hydrophilic domain at each end of the protein. A
single hydrophobic domain spans the whole membrane. Trans-membrane domains tend
to consist of alpha helices Eg Glycophorin (in erythrocytes),
and many other receptors, such as platelet-derived growth factor receptor, insulin receptor,
growth-hormone receptor, etc. (back to figure)

Some are peripheral proteins : they are not inserted in the membrane as
they have no well-defined hydrophobic surface. They are bound to the membrane
principally by ionic associations with the polar phospholipid heads, or with other
membrane proteins. Examples are the profilin, the F1 subunit of ATP-synthase,
and
cytochrome c3cyt (162Kb)[Bbk|BNL|ExP|Waw|Hal]
(back to figure)

The polypeptide chain may traverse the membrane several times. The protein may
have a pore running through it and act as a transmembrane channel or ion pump. Such
proteins have a particular orientation in the membrane: they are functionally
asymmetrical. Examples such as Bacteriorhodopsin are examined
in the next section . (back to figure)

Another type of peripheral protein associates
with the membrane by means of a covalent attachment to a glycolipid in
the bilayer as with some of the immunoglobulin super-family adhesion
molecules, eg Thy-1, NCAM. (back to figure)

Transmembrane domains

The region of a protein which traverses the membrane usually consists
of an alpha-helix (as in type 2 in the figure)
or several alpha-helices ( type 4) each of about 20 residues.
In the latter case, the helices are connected by loops which are exposed to
the aqueous environment on either side of the membrane and which
therefore consist of residues with polar side chains. These connecting
loops are generally quite short and may consist of
hairpins (see Protein Geometry) .

This
diagram shows an example of such a "helix-bundle":
bacteriorhodopsin, a light-driven proton-pump from the purple
membrane of Halobacterium halobium . The colours of the 7
helices are simply to distinguish them in the two perpendicular views.
This is a theoretical model.

Click here 1bac (119Kb)[Bbk|BNL|ExP|Waw|Hal] to examine
the structure in more detail with RasMol. The 'backbone' or 'ribbons'
options from the 'Display' menu best illustrate the arrangement. The
protein has a bound molecule of retinal which can be seen by selecting
'wireframe' mode, etc (select the 'chain' option in the 'Colours'
menu).

Hydropathy of transmembrane helices

In the case of a single helix, mainly hydrophobic residues would be expected,
so that an apolar surface is exposed to the bilayer. This is illustrated in
the diagram (a) below.

If several helices are bundled, then only the side chains on the outside of
the bundle need be hydrophobic. This is illustrated in (b) above. If the
central channel between the helices is lined with polar residues, the resulting
structure might act as a pore in the membrane through which ions may pass.

Membrane proteins are difficult to crystallize. Because they have significant
hydrophilic and hydrophobic surfaces, they are not soluble in aqueous buffer
solutions yet they denature in organic solvents. Methods such as Infra-red
Spectroscopy, Raman Spectroscopy and Circular Dichroism are used to deduce
secondary structure.

However, other approaches may identify transmembrane
regions. Naturally, one would expect a relatively long, uninterrupted sequence
of hydrophobic residues to represent a membrane-spanning structure. On the
other hand, a number of hydrophilic residues would be anticipated in a bundle
structure such as the photosynthetic reaction centre (see also previous diagram
(b). A quantitative measure of the hydrophobicity of a
sequence is required.

Various hydrophobicity scales for amino acids
based on for example the free energy of transfer of the side chain from an
inorganic solvent to water have been compiled. For each
position i in the sequence, a hydropathy index may be calculated as the mean
hydrophobicity value of all the residues from eg i-9 to i+9.
Sharp peaks in a plot of hydropathy index versus residue represent
sequences which would be unusually hydrophobic for a soluble protein and which
therefore are strong candidates for a transmembrane section:

Such plots have in fact been found to be very successful in correctly predicting
membrane-spanning sequences in structures which were subsequently elucidated by
X-ray crystallography, such as the photosynthetic reaction centre.

Photosynthetic Reaction Centre

The photosynthetic reaction centre of the purple bacterium
Rhodopseudomonas occurs in the
membranes of photosynthetic vesicles. This protein complex is composed of
4 subunits: L, M, H and a cytochrome. The L and M subunits are homologous
and each have 5 transmembrane alpha-helices. A helix length of 20-25 residues is
required to span this bacterial membrane. The H subunit, on the cytoplasmic
side of the membrane, also has a single transmembrane helix. As in
bacteriorhodopsin, the helices are tilted at an angle of 20-25 ° to the
perpendicular to the membrane. The whole complex is shown below.Click here 1prc (910Kb) [Bbk|BNL|ExP|Waw|Hal] to look at the
crystal structure in more detail. SCRIPT

A number of pigments (quinones) are bound between the helices of the photosynthetic
reaction centre. Buried helix residues
which interact
either which the pigments, or with other helices are relatively highly conserved
between different bacterial species, whereas those on the outside of the bundle
are not. This indicates the non-specific nature of the hydrophobic interactions
between the complex and the bilayer.

Click
here for another diagram. Also look at the following (thanks to
Manuel Peitsch at GLAXO Geneva):

It should be emphasized however that very few high-resolution crystal
structures
of membrane proteins exist. Other examples to look at are: porin 3por (207Kb)[Bbk|BNL|ExP|Waw|Hal]
(from Rhodobacter ) , which is composed mainly of beta-sheets in a 16-stranded
beta-barrel formation (see section on
all-beta folds )
and forms a
pore in the membrane 1.7 - 2.5 nm in diameter (shown below);
here are
1,2
more images of porin.

Note that the orientation of the strands is such that side chains alternately
point into the interior and exterior of the pore; the former are strongly polar
residues while the latter are very hydrophobic. Here is the
Protein
Science kinemage of the crystal structure. Click
here for
another kinemage.

Also look at the pore-forming domain of colicin A
1col (260Kb)
[Bbk|BNL|ExP|Waw|Hal]. (There
are 2 of these domains in the asymmetric unit of this crystal structure).
Click
here
for a diagram.
Colicin A is an antibiotic which is water-soluble, but which can undergo a
conformational change such that the pore-forming domain inserts itself into
the membrane of E. coli .

If you are able to use MAGE , click
here for the Kinemage supplement to the Branden and Tooze
"Introduction to Protein Structure" chapter on membrane proteins.